Cartilage injuries and arthritis remain an unresolved cause of pain, debility, and diminished quality of life. Our research program goals include developing and testing growth factor and cell-based therapies to improve cartilage healing. Insulin-like growth factor-I (IGF-I) enhances cartilage repair; however, the benefits of IGF-I therapy are tempered by short ligand residence and ongoing pro-inflammatory cytokine flux. We hypothesize that cartilage repair and joint health can be enhanced by a combination of chondrocyte implantation, using cells stably expressing the IGF-I gene, in concert with a less hostile environment developed by silencing IL-1b. Adeno-associated virus (AAV) will be used to enhance transgene longevity in chondrocyte-transplanted cartilage defects in a large animal model. Concurrently, IL-1 message will be knocked down using RNA interference, which will limit cartilage degradation and pain through reduced metalloprotease (MMP) and aggrecanase proliferation. The hypothesis will be tested by these project aims: 1) Evaluate cartilage repair, cell survival, and transgene persistence in large full-thickness cartilage defects grafted with chondrocytes over expressing IGF-I. Proof of principle shows adenovirus-IGF-I enhances early cartilage repair; AAV should extend and enhance this effect long-term. 2) Evaluate IL-1b gene silencing using RNA interference motifs to moderate articular cytokine fluxes in chondrocyte and synovial cell cultures. Preliminary data shows RNA interference cassettes with perfect nucleotide identity to target IL-1 mRNA silence cognate IL-1 expression. These experiments screen the transduction efficiency and determine downstream signaling effects of small interfering RNAs. 3) Assess the effects of IL-1b knockdown resulting from synoviocyte transduction with siRNAs on the restoration of arthritic cartilage matrix quality in synoviocyte/ cartilage co-cultures. Synoviocytes actively synthesize IL-1 and are ideal primary targets for IL- 1b silencing. Subsequent effects on degraded cartilage in this culture model of arthritis should reflect the ability of IL-1b knockdown to improve arthritic cartilage matrix. 4) Extend siRNA effects by developing stably integrating plasmid vectors. In vivo application will require episomal or integrated plasmid DNA delivery and these studies test these motifs. 5) Assess enhanced cartilage repair from transplantation of IGF-I gene transduced chondrocytes to joints where catabolic activity is reduced by locally expressing IL-1b knockdown elements. Combined, the results of these 5 aims should assess anabolic gene enhanced cartilage repair in an environment made permissive to long-term and durable repair by IL-1 mRNA silencing. This is the first time AAV-IGF-I will be applied in a cartilage repair mode, and additional benefits are expected using RNA interference, which has recently been described in a therapeutic role in medicine. Application of these technologies to cartilage repair, and potentially early stages of arthritis, provide significant hope for control of the progression of arthritis and potentially minimize the consequences of joint injury. PUBLIC HEALTH RELEVANCE. Cartilage injuries and arthritis remain an unresolved cause of pain, debility, and diminished quality of life. Our research program goals include developing and testing growth factor and cell- based therapies to improve cartilage healing. Insulin-like growth factor-I (IGF-I) enhances cartilage repair and this grant tests long-term growth factor expression in joints where degradation is controlled by RNA interference.